Project Summary/Abstract Although antiretroviral therapy (ART) inhibits HIV replication and decreases morbidity and mortality, it does not cure. Interruption of ART is routinely followed by viral rebound springing from a small but durable reservoir of latently infected, long lived, memory CD4 T cells. New chemical-, immune- and gene-and cell- based therapies are now being developed that will be aimed at eradicating this reservoir (difficult to achieve) or reducing its size and boosting boosting antiviral immunity sufficiently that ART can be safely stopped without high level viral rebound (functional cure). The search for curative therapies would be greatly facilitated by the availability of a set of robust biomarkers that can accurately predict whether a specific therapeutic is effective or not. Such biomarkers could help ensure that only the most promising candidates advance to analytic treatment interruption??the current gold standard for clinical testing of cure therapeutics. ATI studies need to be minimized because they are inherently costly, logistically challenging and create potential risks for patients during viral rebound. These biomarkers might also provide key insight into what biological processes are most promising for attacking the reservoir and achieving the desired delay in time to rebound. We hypothesize that highly robust virus-specific and host?specific biomarkers can be identified in blood that accurately predict the duration of viral remission after treatment interruption as well as impending viral rebound. To pursue identification of both virus- and host-directed biomarkers, blood samples from 125 HIV infected subjects (both treated during acute and chronic infection) obtained before ATI and after viral rebound will be analyzed by three different approaches: (1) Resting blood CD4 T cells will be tested with a novel digital droplet PCR assay (IPDA) that selectively detects intact proviral DNA in the reservoir??these intact viruses represent the key small fraction of the proviruses in the reservoir that are replication competent and thus able to drive viral rebound. Low IPDA results prior to ATI could be associated with long times to viral rebound; this assay can be performed in 6 hours and only requires the equivalent of a 20 ml blood draw; (2) RNA from both CD4 T and non-CD4 T cells will be subjected to RNA-Seq and miRNA-Seq analyses to identify patterns of gene expression associated with markedly delayed or accelerated times to viral rebound and (3) Measurement of pyroptotic inflammatory markers in cells and plasma as biomarkers predicting rapid loss of viral control or impending viral rebound during ATI. The complex data sets generated by RNA-Seq will analyzed with the help of the Bioinformatics and Biostatistics core. This project will also closely interface with both Projects 1 and 2 where complementary approaches will be pursued searching for biomarkers in blood cells and in plasma or circulating extracellular vesicles. By careful execution of this comprehensive virus- and host-directed search, our project team should be strongly positioned to discover a robust set of new biomarkers that could truly galvanize future HIV cure research.